During illumination, the light-sensitive plasma membrane (rhabdomere) of Drosophila photoreceptors undergoes turnover with consequent changes in size and composition. However, the mechanism by which illumination is coupled to rhabdomere turnover remains unclear. We find that photoreceptors contain a light-dependent phospholipase D (PLD) activity. During illumination, loss of PLD resulted in an enhanced reduction in rhabdomere size, accumulation of Rab7 positive, rhodopsin1-containing vesicles (RLVs) in the cell body and reduced rhodopsin protein. These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD. In wild-type photoreceptors, during illumination, enhanced PLD activity was sufficient to clear RLVs from the cell body by a process dependent on Arf1-GTP levels and retromer complex function. Thus, during illumination, PLD activity couples endocytosis of RLVs with their recycling to the plasma membrane thus maintaining plasma membrane size and composition.DOI: http://dx.doi.org/10.7554/eLife.18515.001

The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning "grapes"). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to s 2 -adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the s 2 -adaptin mutation. Genetic perturbation of s 2 -adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38°. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor b (TGFb) signaling was altered in these mutants and was restored by normalizing s 2 -adaptin in neurons. Thus, our data suggest that (1) while s 2 -adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during highfrequency nerve stimulation, and (2) s 2 -adaptin regulates NMJ morphology by attenuating TGFb signaling. KEYWORDS Drosophila; angur; synapse; physiology; pMAD S YNAPTIC transmission requires fusion of synaptic vesicles (SVs) at the active zones followed by their efficient retrieval and recycling through endocytic mechanisms (Heuser and Reese 1973;Jahn and Sudhof 1994). Retrieval and sorting of membrane lipids and vesicular proteins at the synapse are mediated by a well-orchestrated and coordinated action of several adapter and endocytic proteins (Stimson et al. 2001;Rikhy et al. 2002;Verstreken et al. 2002;Koh et al. 2004;Marie et al. 2004). Clathrin-mediated endocytosis (CME) is the primary pathway operative at the synapses for membrane retrieval (Granseth et al. 2006(Granseth et al. , 2007Heerssen et al. 2008;Dittman and Ryan 2009;McMahon and Boucrot 2011;Saheki and De Camilli 2012). Genetic analysis of the components of the CME pathway in Caenorhabditis elegans and Drosophila has revealed that this pathway is required for SV re-formation, and in many cases, blocking CME at synapses results in temperature-sensitive paralysis (Gonzalez-Gaitan and Jackle 1997;Zhang et al. 1998;Stimson et al. 2001;Koh et al. 2004Koh et al. , 2007Sato et al. 2009). Additionally, CME plays a crucial role in regulating synaptic morphology (Rikhy et al. 2002;Koh et al. 2004Koh et al. , 2007Dickman et al. 2006). At Drosophila NMJs, blocking CME results in enhanced bone morphogenetic protein (BMP) si...

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The activation of phospholipase C (PLC) is a conserved mechanism of receptor-activated cell signaling at the plasma membrane. PLC hydrolyzes the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P], and continued signaling requires the resynthesis and availability of PI(4,5)P at the plasma membrane. PI(4,5)P is synthesized by the phosphorylation of phosphatidylinositol 4-phosphate (PI4P). Thus, a continuous supply of PI4P is essential to support ongoing PLC signaling. While the enzyme PI4KA has been identified as performing this function in cultured mammalian cells, its function in the context of an physiological model has not been established. In this study, we show that, in photoreceptors, PI4KIIIα activity is required to support signaling during G-protein-coupled PLC activation. Depletion of PI4KIIIα results in impaired electrical responses to light, and reduced plasma membrane levels of PI4P and PI(4,5)P Depletion of the conserved proteins Efr3 and TTC7 [also known as StmA and L(2)k14710, respectively, in flies], which assemble PI4KIIIα at the plasma membrane, also results in an impaired light response and reduced plasma membrane PI4P and PI(4,5)P levels. Thus, PI4KIIIα activity at the plasma membrane generates PI4P and supports PI(4,5)P levels during receptor activated PLC signaling.